The discovery of small molecules capable of selectively modulating the activity of biological targets is a central challenge of chemical biolog that has resulted in many valuable human therapeutics and research tools. At the start of the original granting period eight years ago, our laboratory initiated a program to develop a new approach to the synthesis and discovery of bioactive small molecules that combines powerful aspects of biosynthesis and biological evolution with synthetic organic chemistry. We discovered that DNA duplex formation exerts remarkable control over the effective molarity of DNA- linked reactants, including those unrelated in structure to DNA. The resulting development of DNA- templated organic synthesis has enabled DNA sequences encoding synthetic molecules to undergo translation, selection, and amplification that parallel key steps in biologica evolution. During the original granting period, we developed the foundations of DNA-templated synthesis, developed in vitro selections of DNA-linked small molecules, and developed and applied a novel DNA- encoded approach to the discovery of bond-forming reactions. In the second granting period (spanning the past four years), we performed the first large-scale DNA-templated library synthesis, subjected the resulting DNA-templated macrocycles to a broad set of in vitro selections resulting in the discovery of several new families of inhibitors of disease-associated targets, characterized in depth (including two X- ray co-crystal structures) the mechanism of action of a class of highly selective kinase inhibitors emerging from these selections, developed a second-generation DNA-encoded reaction discovery system, and used this system to discover two new reactions. We also developed two new methods, reactivity-dependent PCR (RDPCR) and interaction-dependent PCR (IDPCR) that transduce binding or bonding events into the selective amplification of DNA sequences encoding desired molecules. Here we propose to characterize and develop the in vitro and in vivo properties of a family of potent, selective, and allosteric macrocyclic inhibitors of insulin-degrading enzyme (IDE) that we recently discovered from the in vitro selection of a 13,284-membered DNA-templated macrocycle library. These inhibitors will enable us to experimentally answer for the first time the longstanding question of whether or not acute IDE inhibition in vivo has potential as a therapeutic strategy for diabetes. In addition, we will build on the promising features of IDPCR to develop a new capability that enables DNA-linked libraries to be evaluated for their ability to interact with target proteins in the more physiologically relevant context of cell lysates, rather than in purified form. Finally, we will broadly apply this new methodology to the discovery of new DNA-templated and DNA-conjugated small molecules with the ability to modulate the activities of therapeutically relevant targets. PUBLIC HEALTH RELEVANCE: We have developed a novel approach to the discovery of bioactive small molecules that has led to new inhibitors for disease-associated proteins including certain kinase and protease enzymes. We propose to develop one class of these molecules to reveal the potential of insulin-degrading enzyme as a target for treating diabetes and as a starting point for the development of future anti-diabetes medicines. We also propose the development of new methods that have the potential to greatly improve the efficiency and biological relevance of the small-molecule discovery process.